Analysis Note

Positive ControlRaw 264.7 cells treated with IFNγ and LPS

Application

Frozen Sections (1:500)r>Immunoblotting (1:2000)r>Immunocytochemistry (1:500)r>Paraffin Sections (1:500)

General description

This Anti-Nitric Oxide Synthase, Inducible Rabbit pAb is validated for use in Frozen Sections, WB, ICC, Paraffin Sections for the detection of Nitric Oxide Synthase, Inducible.

Rabbit polyclonal antibody supplied as undiluted serum. Recognizes the ~130 kDa inducible nitric oxide synthase (iNOS) protein.

Recognizes the ~130 kDa iNOS protein.

Immunogen

a synthetic peptide (CKKGSALEERKATRL) corresponding to amino acids 1131-1144 of mouse macrophage inducible nitric oxide synthase (iNOS), conjugated to KLH

Mouse

Legal Information

CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Exhibits no cross-reactivity with bNOS or eNOS. Variables associated with assay conditions will dictate the proper working dilution.r>r>Recommended Procedure for Immunocytochemistry of Anti-iNOS:r>1. Grow cells in appropriate medium.r>2. Induce NOS by pretreating cells for 16 h with fresh media containing either: no addition (control), interferon-γ (50 ng/ml), LPS (30 µg/ml) or interferon plus LPS.r>3. Wash cells 3-5 min with phosphate buffered saline (PBS).r>4. Permeabilize with 2% saponin for 10 min at room temperature.r>5. Fix in 4% formalin for 10 min at room temperature.r>6. Wash 3-5 min with PBS.r>7. Incubate for 20 min at room temperature with anti-iNOS diluted 1:500 in PBS.r>8. Wash 3-5 min with PBS.r>9. Incubate with biotinylated secondary antibody (i.e. biotin-conjugated goat anti-rabbit IgG, Cat. No. 401313). Wash; incubate with avidin-conjugated alkaline phosphatase (Cat. No. 189726 or 189732).r>10. Develop with Naphthol AS Phosphate/Fast Red Violet LB (Cat. No. 477758) or Naphthol AS Phosphate/New Fuchsin (Cat. No. 477759).

Lowenstein, C., et al. 1992. Proc. Natl. Acad. Sci. USA89, 6711.Lyons, C., et al. 1992. J. Biol. Chem.267, 6370.Bredt, D.S., et al. 1991. Nature351, 714.

Packaging

100 µL in Plastic ampoule

Physical form

Undiluted serum.

Reconstitution

Following initial thaw, aliquot and freeze (-20°C).

Warning

Toxicity: Standard Handling (A)